Abstract

De novo mutations are recognized both as an important source of genetic variation and as a prominent cause of sporadic disease in humans. Mutations identified as de novo are generally assumed to have occurred during gametogenesis and, consequently, to be present as germline events in an individual. Because Sanger sequencing does not provide the sensitivity to reliably distinguish somatic from germline mutations, the proportion of de novo mutations that occur somatically rather than in the germline remains largely unknown. To determine the contribution of post-zygotic events to de novo mutations, we analyzed a set of 107 de novo mutations in 50 parent-offspring trios. Using four different sequencing techniques, we found that 7 (6.5%) of these presumed germline de novo mutations were in fact present as mosaic mutations in the blood of the offspring and were therefore likely to have occurred post-zygotically. Furthermore, genome-wide analysis of "de novo" variants in the proband led to the identification of 4/4,081 variants that were also detectable in the blood of one of the parents, implying parental mosaicism as the origin of these variants. Thus, our results show that an important fraction of de novo mutations presumed to be germline in fact occurred either post-zygotically in the offspring or were inherited as a consequence of low-level mosaicism in one of the parents.

Workflow for the Detection of Mosaic Mutations among a Subset of Apparently De Novo Mutations(A) Assessment of technique-dependent variation in sequencing of two groups of heterozygous germline variants (in blue) for determining the distribution of allelic ratios for three different techniques (WGS, ADS, and Sanger sequencing).(B) Previously identified de novo mutations were re-sequenced by ADS and Sanger sequencing for determining the variant ratio. With the use of the combined Z score, nine putative somatic variations were identified. They were then validated by ADS with a second independent primer pair and smMIPs. Seven of nine were confirmed to deviate in allelic ratio, suggesting a non-germline event.(C) Identification of de novo mutations originating from parental mosaicism. Of 4,081 high-confidence de novo mutations identified by WGS, 13 were identified to have two or more variant reads in parental DNA. With the use of ADS data from the non-carrier parent for correcting for the background sequencing error, four mutations appearing as de novo in the child were identified as low-level mosaicism in one of the parents.